• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Nov 2010; 192(21): 5848–5849.
Published online Aug 27, 2010. doi:  10.1128/JB.00951-10
PMCID: PMC2953705

Complete Genome Sequence of Staphylococcus aureus Strain JKD6008, an ST239 Clone of Methicillin-Resistant Staphylococcus aureus with Intermediate-Level Vancomycin Resistance[down-pointing small open triangle]

Abstract

We report here the complete 2.92-Mb genome sequence of a clinical isolate of methicillin-resistant Staphylococcus aureus subsp. aureus that demonstrates intermediate-level vancomycin resistance. The strain, named JKD6008, belongs to multilocus sequence type 239 and was isolated from the bloodstream of a patient in New Zealand in 2003.

We have previously described the in vivo evolution of low-level vancomycin resistance in Staphylococcus aureus through comparative and functional genomic assessment of a pair of isogenic methicillin-resistant Staphylococcus aureus (MRSA) strains. The vancomycin-susceptible S. aureus (VSSA) strain JKD6009 was a patient wound isolate, whereas vancomycin-intermediate S. aureus (VISA) strain JKD6008 was recovered from the bloodstream of the same patient after 42 days of vancomycin treatment (5). Comparison of the partially assembled genomes of the two isolates revealed a single-point mutation in the sensor region of the two-component regulatory gene graS, which caused a significant reduction in the vancomycin susceptibility of JKD6008 (6). Here we report the fully assembled and annotated genome of S. aureus JKD6008.

The genome sequence of S. aureus strain JKD6008 was determined by whole-genome shotgun sequencing using single-read 454 GS20 (Roche Diagnostics, Basel, Switzerland), Sanger (Applied Biosystems), and SOLiD (Applied Biosystems) sequencing technologies, producing approximately 20 times, 4 times, and 225 times coverage of the genome, respectively. GS20 reads were assembled using gsAssembler v2.0 software, resulting in 131 contigs (≥500 bp) totaling 2.83 Mbp (6, 10). Sanger paired-end reads (clone insert size, 3 to 5 kb) were combined with the GS20 contigs using Gap4 v4.11 software (3). Mate-pair SOLiD reads (3 to 5 kb) were aligned to the contigs using SHRiMP 1.3.2 software to identify and correct sequencing errors (11). Optical mapping produced a high-resolution XbaI chromosome restriction map, to which the contigs were aligned using MapSolver 2.1.1 (Opgen) to determine misassemblies. Gap closures were performed by PCR, followed by Sanger sequencing and primer walking of amplification products (3730S DNA Analyzer sequencer; Applied Biosystems). The assembly of the completed genome was confirmed to be correct by reference to the XbaI optical map.

Protein-coding regions were predicted using GeneMarkS 4.6b software, tRNA genes using tRNAscan-SE 1.23, and rRNA genes using RNAmmer 1.2 (2, 8, 9). Gene products were assigned using HMMER 3.0 against the Pfam database (release 23) and BLAST 2.2.23 against RefSeq proteins (April 2010) and the Conserved Domain Database (v2.22) (1, 4). These automated analyses were followed by manual curation and comparisons with other completed S. aureus genomes.

The genome of S. aureus strain JKD6008 consists of a circular 2,924,344-bp chromosome with a 34% G+C content and no extrachromosomal elements. A total of 2,766 coding DNA sequences, 82 tRNA genes, and 5 rRNA loci were detected. Over 70% of genes were assigned to specific Clusters of Orthologous Groups (COG) functional groups, and 42% were assigned an enzyme classification number (12).

Initial analysis of the whole-genome sequence of JKD6008 confirmed it as a member of the ST239 complex, sharing 2,504 orthologous coding sequences (CDSs) with the recently described ST239 member TW20 (EMBL accession no. FN433596.1). There are 17 copies of IS256 and a type III staphylococcal cassette chromosome mec element (SCCmec). Comparisons with 19 published S. aureus genomes revealed 20 CDS not present in any other S. aureus genome, although some of these 20 CDS have orthologs in other Staphylococcus species. JKD6008 also harbors a 28-kb integrated pSK1-like plasmid that is predicted to confer resistance to aminoglycosides and trimethoprim, as well as efflux-mediated antiseptic and disinfectant resistance (7).

Nucleotide sequence accession number. The complete genome sequence has been deposited in NCBI GenBank under accession number CP002120.

Acknowledgments

This work was supported by the National Health and Medical Research Council of Australia and the Austin Hospital Medical Research Foundation.

Footnotes

[down-pointing small open triangle]Published ahead of print on 27 August 2010.

REFERENCES

1. Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402. [PMC free article] [PubMed]
2. Besemer, J., A. Lomsadze, and M. Borodovsky. 2001. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res. 29:2607-2618. [PMC free article] [PubMed]
3. Bonfield, J. K., K. F. Smith, and R. Staden. 1995. A new DNA sequence assembly program. Nucleic Acids Res. 23:4992-4999. [PMC free article] [PubMed]
4. Eddy, S. R. 2009. A new generation of homology search tools based on probabilistic inference. Genome Inform. 23:205-211. [PubMed]
5. Howden, B. P., P. D. Johnson, P. B. Ward, T. P. Stinear, and J. K. Davies. 2006. Isolates with low-level vancomycin resistance associated with persistent methicillin-resistant Staphylococcus aureus bacteremia. Antimicrob. Agents Chemother. 50:3039-3047. [PMC free article] [PubMed]
6. Howden, B. P., T. P. Stinear, D. L. Allen, P. D. Johnson, P. B. Ward, and J. K. Davies. 2008. Genomic analysis reveals a point mutation in the two-component sensor gene graS that leads to intermediate vancomycin resistance in clinical Staphylococcus aureus. Antimicrob. Agents Chemother. 52:3755-3762. [PMC free article] [PubMed]
7. Jensen, S. O., S. Apisiridej, S. M. Kwong, Y. H. Yang, R. A. Skurray, and N. Firth. 12 June 2010, posting date. Analysis of the prototypical Staphylococcus aureus multiresistance plasmid pSK1. Plasmid [Epub ahead of print.] doi:.10.1016/j.plasmid.2010.06.001 [PubMed] [Cross Ref]
8. Lagesen, K., P. Hallin, E. A. Rødland, H. H. Staerfeldt, T. Rognes, and D. W. Ussery. 2007. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 35:3100-3108. [PMC free article] [PubMed]
9. Lowe, T. M., and S. R. Eddy. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25:955-964. [PMC free article] [PubMed]
10. Margulies, M., M. Egholm, W. E. Altman, S. Attiya, J. S. Bader, L. A. Bemben, J. Berka, M. S. Braverman, Y. J. Chen, Z. Chen, S. B. Dewell, L. Du, J. M. Fierro, X. V. Gomes, B. C. Godwin, W. He, S. Helgesen, C. H. Ho, G. P. Irzyk, S. C. Jando, M. L. Alenquer, T. P. Jarvie, K. B. Jirage, J. B. Kim, J. R. Knight, J. R. Lanza, J. H. Leamon, S. M. Lefkowitz, M. Lei, J. Li, K. L. Lohman, H. Lu, V. B. Makhijani, K. E. McDade, M. P. McKenna, E. W. Myers, E. Nickerson, J. R. Nobile, R. Plant, B. P. Puc, M. T. Ronan, G. T. Roth, G. J. Sarkis, J. F. Simons, J. W. Simpson, M. Srinivasan, K. R. Tartaro, A. Tomasz, K. A. Vogt, G. A. Volkmer, S. H. Wang, Y. Wang, M. P. Weiner, P. Yu, R. F. Begley, and J. M. Rothberg. 2005. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437:376-380. [PMC free article] [PubMed]
11. Rumble, S. M., P. Lacroute, A. V. Dalca, M. Fiume, A. Sidow, and M. Brudno. 22 May 2009. SHRiMP: accurate mapping of short color-space reads. PLoS Comput. Biol. 5:e1000386. [Epub ahead of print.] [PMC free article] [PubMed]
12. Tatusov, R. L., N. D. Fedorova, J. D. Jackson, A. R. Jacobs, B. Kiryutin, E. V. Koonin, D. M. Krylov, R. Mazumder, S. L. Mekhedov, A. N. Nikolskaya, B. S. Rao, S. Smirnov, A. V. Sverdlov, S. Vasudevan, Y. I. Wolf, J. J. Yin, and D. A. Natale. 2003. The COG database: an updated version includes eukaryotes. BMC Bioinformatics 4:41. [PMC free article] [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...